Clinico-pathological significance of extra-nodal spread in special types of breast cancer

Objective To investigate the significance of extra-nodal spread in special histological sub-types of breast cancer and the relationship of such spread with prognostic parameters. Methods A total of 303 breast cancer cases were classified according to tumor type, and each tumor group was subdivided according to age, tumor diameter, lymph node metastasis, extra-nodal spread, vein invasion in the adjacent soft tissue, distant metastasis, and immunohistochemical characteristics [estrogen receptor (ER), progesterone receptor (PR) existence, p53, c-erbB-2, and proliferative rate (Ki-67)]. The 122 cases with extra-nodal spread were clinically followed up. Results An extra-nodal spread was observed in 40% (122 cases) of the 303 breast cancer cases. The spread most frequently presented in micro papillary carcinoma histological sub-type (40 cases, 75%), but least frequently presents in mucinous carcinoma (2 cases, 8%). Patients with extra-nodal spread had a high average number of metastatic lymph nodes (8.3) and a high distant metastasis rate (38 cases, 31%) compared with patients without extra-nodal spread. Conclusion The existence of extra-nodal spread in the examined breast cancer sub-types has predictive value in forecasting the number of metastatic lymph nodes and the disease prognosis.


Introduction
Breast carcinoma is the most common malignant tumor and is the leading cause of cancer death in women, with an incidence of more than 90 new cases per pollution of population 100,000 annually. Breast cancer has specific histopathological types with different prognostic and clinical characteristics. Histological type, age, tumor diameter, existing lymph node metastasis, the number of the metastatic lymph nodes, the expression of estrogen receptor (ER) and progesterone receptor (PR), proliferative rate (Ki-67), p53 mutation, and c-erbB-2 are oncogenic-prognostic determinants of breast carcinoma [1][2][3] . Both the presence and the total number of tumor-positive lymph nodes are of prognostic value. Grouping patients with one to three, four to nine, and ten or more positive lymph nodes metastases is a generally accepted approach and is used to determine the type of chemotherapy to be given after surgery. Previous studies [4][5][6][7][8] assert that in the identification of prognosis, extra-nodal spread has a significant relationship with complete survival, disease-free survival, and local recurrence. This study examines the significance of extranodal spread in breast cancer cases and its relationship with prognostic parameters. The significance of the extra-nodal spread in special histological sub-types of breast cancer, as well as its predictive value in guiding treatment, is discussed.

Materials and methods
A total of 303 cases diagnosed between 1993 and 2004 were selected for the study. The study was approved by the institutional ethics committee. All patients underwent mastectomy and axillary dissection. A total of 100 cases were diagnosed with invasive ductal carcinoma (IDC), 60 with invasive lobular carcinoma (ILC), 37 with pleomorphic lobular carcinoma (PLC), 25 with mucinous carcinoma, 28 with mixed mucinous carcinoma, and 53 with micropapillary carcinoma. After the 303 cases were classified according to tumor type, each tumor group was subdivided according to age, tumor diameter, lymph node metastasis, extranodal spread, vascular invasion in the adjacent soft tissue, distant metastasis, and immunohistochemical characteristics.

Immunohistochemistry
Tumor specimens were fixed in 10% neutral buffered formalin for 20 to 28 h before processing and embedding. Sections were cut on a microtome and stained with H&E. ER, PR, c-erbB-2, and Ki67 were determined through immunohistochemistry by using a streptavidine biotine peroxidase system. Staining protocols for each antibody had previously been optimized and standardized. First, 4 mm thick tissue sections were cut from representative blocks and mounted on charged and pre-cleaned slides. Second, sections were dewaxed in xylene and rehydrated through a decreasing concentration of alcohol to water. Then, heat-induced antigen retrieval (immersion in citrate buffer, pH 6.0, at 95 °C for 30 min followed by cooling at room temperature for 20 min) or, alternatively, a 10 min treatment with protease XIV was applied. Sections were incubated with 0.3% hydrogen peroxide in methanol for 30 min. to block endogenous peroxidase activity. Immunostaining experiments for the localization of ER and PR, p53, c-erbB-2 protein, and Ki-67 antigen were conducted on consecutive tissue sections from the tumor-containing blocks. The primary antibodies used were the monoclonal antibody (Mab) to ER (clone 1D5, at 1/100 dilution; Dako, Glostrup, Denmark), the Mab to PGR (clone 1A6, at 1/800 dilution; Dako), the MIB-1 Mab to the Ki-67 antigen (at 1/1,200 dilution; Immunotech, Marseille, France), and the polyclonal antibody (at 1/3,200 dilution; Dako) to the c-erbB-2 protein.
The immunohistochemically stained slides were evaluated by three pathologists who were all very experienced in breast pathology. Observers were asked to select at least three highpower fields to comprise the spectrum of staining seen on the initial overview. The average percentage of positive stained cells across the section were scored. Only nuclear reactivity, irrespective of its intensity, was considered for ER ( Figure  1), PR , p53, and Ki-67 antigens, whereas only an intense and complete membrane staining in >10% of the tumor cells qualified for c-erbB-2 overexpression (3+) (Figure 2). Steroid hormone receptors status and p53 were classified as negative in case of <1% immunoreactive tumor cells ( Table 1). The scoring of c-erbB-2 by immunohistochemistry was semiquantitatively evaluated according to the following categories: 0, no membrane staining; 1+, weak and incomplete membrane staining; 2+, weak/moderate heterogeneous complete membrane staining in <10% of tumor cells; or 3+, strong, complete, homogeneous membrane staining in >10% of tumor cells.

Clinical follow-up of 122 cases with extra-nodal spread Examined data
• Age: under 50 and above 50; • Lymph node metastasis: no axillary metastasis, metastasis in one to three lymph nodes, metastasis in four to nine lymph nodes, and metastasis in 10 or more lymph nodes;

Statistical analysis
Statistical analysis was performed by using the statistical analysis system. Chi-square and t-tests were used. The cases with P values under 0.05 were considered significant.

Invasive ductal carcinoma (IDC)
The patients with extra-nodal spread had a significantly higher mean number of metastatic lymph node (8.6) and distant metastasis ratio (10 cases, 10%) compared with the patients without extra-nodal spread (P<0.05). A significant relationship exists between the extra-nodal spread presence and four or more lymph node metastases (P<0.05) (Figure 3).

Invasive lobular carcinoma (ILC)
The patients with extra-nodal spread had significantly higher mean number of metastatic lymph nodes (12.3) and distant metastasis ratio (5 cases, 8.3%) compared with the patients without extra-nodal spread (P<0.05). A significant relationship exists between the extra-nodal spread existence and ten or more lymph nodes metastasis (P<0.05). C-erbB-2 was highly positive in patients with extra-nodal spread (66%) compared with patients without such spread (44%) (P<0.05).

Pleomorphic lobular carcinoma (PLC)
The patients with extra-nodal spread had a significantly higher mean tumor size (2.2 cm), mean number of metastatic lymph nodes (11), and distant metastasis ratio (5 cases, 13%) compared with the patients without extra-nodal spread (P<0.05). A significant relationship exists between extra-nodal spread and the presence

Micropapillary carcinoma
Patients with extra-nodal spread had a significantly higher mean number of metastatic lymph node (9.7) and distant metastasis ratio (14 cases, 26%) compared with the patients without extranodal spread (P<0.05). A significant relationship exists between the extra-nodal spread and the existence of four or more lymph nodes metastasis (P<0.05). The number of the lymph node metastasis was higher in the axilla in micropapillary carcinoma compared with the cases diagnosed with mucinous and mixed mucinous carcinoma (P<0.05).

Mucinous carcinoma
Patients with extra-nodal spread had a significantly higher mean number of metastatic lymph node (5.5) and the distant metastasis ratio (2 cases, 8%) compared with the patients without extra-nodal spread (P<0.05). In the mucinous carcinoma cases with extra-nodal spread, p53 was significantly more positive than that in cases without extra-nodal spread (P<0.05). The cases diagnosed with mucinous carcinoma had the least ratio (8%) of extra-nodal spread among all groups (P<0.05). Compared with IDC, mucinous carcinoma was observed in the older population (P<0.05).

Mixed mucinous carcinoma
Patients with extra-nodal spread had a significantly higher mean number of metastatic lymph node (2.7%) and distant metastasis ratio (2 cases, 7%) compared with the patients without extra-nodal spread (P<0.05). A significant relationship exists between extra-nodal spread and the existence of 4-9 lymph node metastases (P<0.05). C-erbB-2 was highly positive in the mixed mucinous tumors with extra-nodal spread, whereas p53 was interestingly high in patients without extra-nodal spread (P<0.05).

Considering all the cases together as a whole (Table 2)
Extra-nodal spread was found in 40% (122 cases) of 303 breast cancer cases. While the extra-nodal spread most frequently presents in the histological sub-type of micropapillary carcinoma (40 cases, 75%), it least frequently presents in mucinous carcinoma (2 cases, 8%).
Patients with extra-nodal spreads had higher mean number of metastatic lymph nodes (8.3) and distant metastasis ratio (28 cases, 9%) compared with the patients without extra-nodal spread.
A relationship exists between the extra-nodal spread and the presence of four or more lymph node metastases (P<0.05), as well as between the number of metastatic lymph nodes (≥4) and tumor diameter, extra-nodal spread, distant metastasis, and extra-nodal vascular invasion presence (P<0.05). Apart from the tumor type, the presence of distant metastasis was associated with extra-nodal spread.
The relationship between the extra-nodal vascular invasion and the distant metastasis was investigated. However, the results had a limit value (P=0.061).

Discussion
One of the most important prognostic factors in breast cancer is the regional lymph node status 9 . Previous studies [10][11][12][13] observed that the extra-nodal spread occurred with disseminated axillary lymph node metastases. Vicini et al. 13 proved that patients with extra-nodal spread had significantly more mean metastatic lymph nodes. The mean number of metastatic lymph nodes of the Patients were divided into three groups according to the number of metastatic lymph nodes (1-3, 4-9, ≥10). A significant relationship was found between extra-nodal spread and the presence of four or more lymph nodes metastases (P<0.05) in the IDC, micropapillary, and mixed mucinous carcinoma groups. The ILC, PLC, and mucinous carcinoma groups exhibited a significant relationship between extra-nodal spread and the presence of ten or more lymph node metastases (P<0.05). Previous studies [10][11][12][13] emphasized that the number of metastatic axillary lymph nodes was four or more in patients with extranodal spread. The exclusion of extra-nodal invasion by recent TNM versions seems reasonable, mainly because of its direct association with the number of positive lymph nodes and the lack of independent prognostic significance.
Although Leonard et al. 11 , Fisher et al. 14 ,and Mignano et al. 4 stated that the extra-nodal spread affected survival, whereas Donegan et al. 10 and Pierce et al. 12 suggested that it was not. The results of our study show a significant relationship between distant metastases and extra-nodal spread (P<0.05). Notably, distant metastases were observed in 31% of the patients with extra-nodal spread but in only 7% of the patients without extranodal spread.
A significant relationship between the tumor diameter and the extra-nodal spread was only observed in the PLC group (P<0.05).
However, a significant relationship between tumor diameter and the presence of four or more metastatic lymph nodes was observed in all groups, which suggests that extra-nodal spread might have an indirect relationship with the tumor diameter, which is a prognostic factor.
The cases were also related to the presence of vascular invasion on the extra-nodal area in patients. Vascular invasion on adjacent soft tissue was observed in 20% of all patients with extra-nodal spread (61 cases), 14% in IDC (14 cases), 8.5% in ILC (5 cases), 7.1% in PLC (2 cases), 45% in micropapillary carcinoma (23 cases), 4% in mucinous carcinoma (1 case), and 7% in mixed mucinous carcinoma (2 cases) ( Table 2). The results were significantly when compared with the findings on patients without extra-nodal spread (no extra-nodal vascular invasion was observed) (P<0.05). The relationship between extra-nodal vein invasion and distant metastases was also researched, but the results had a limit value (P=0.061).
In this study, no axillary recurrence was observed. Previous studies observed axillary recurrence to be 0% (9) or at a very low level of 3% (4 and 10) in cases with extra-nodal spread and no radiotherapy.
Local recurrence was observed in only four (10%) (two IDC, one PLC, one micropapillary carcinoma) of 38 cases that received partial mastectomy. Bucci et al. 5 also failed to detect local recurrence in any of their 43 patients with extra-nodal spread who received adjuvant radiotherapy. Certainly, the low local recurrence ratio in our cases can be attributed to the adjuvant radiotherapy used on all patients. The current findings show that the presence of extra-nodal spread in the examined breast cancer sub-types has predictive value in forecasting the number of metastatic lymph nodes and in disease prognosis.